In the past year, we have conducted the following studies in the chimpanzee model of hepatitis C virus infection: (i) Identification and characterization of novel chimpanzee MHC class I and class II alleles T cells recognize viral peptides in the context of MHC class I and II molecules on the surface of cells that are either infected by the virus or take up and process exogenous viral antigens. Chimpanzee (Pan troglodytes) MHC alleles have been designated Patr alleles. Here, we have continued our efforts to identify and characterize (i) the Patr class I and II alleles of chimpanzees and (ii) the HCV peptides that they present. In a collaboration with extramural investigators, we identified twelve new Patr class I alleles and two new Patr-DR sequences to date, which have been submitted to the IPD/MHC-NHP database (Robinson et al., Nucleic Acids Research 2003: 31:311-14) and to GenBank. Further, we have identified a panel of specific HCV epitopes that are presented by these Patr alleles. In in vitro studies using T cells from chimpanzees with acute HCV infection we identified the minimal optimal sequence and Patr-restriction of these epitopes. The identification and characterization of Patr alleles and Patr-binding viral peptides is important for the generation of immunological tools such as Patr-peptide tetramers which can then be used to characterize effective cellular immune responses ex vivo in liver biopsies and blood samples throughout the course of HCV infection in this animal model. (ii) Characterization of the kinetics of HCV-specific immune responses in blood and liver in the early and acute phase of HCV infection The generated tetramers were subsequently used to stain HCV-specific T cells throughout the course of HCV infection in chimpanzees. In chimpanzees and humans the onset of acute hepatitis with increased alanine aminotransferase levels is typically observed not earlier than 8-10 weeks after infection and coincides with the accumulation of T cells in the liver. This apparent delay of adaptive immune responses is striking because HCV titers increase to up to 10e7 to 10e9 genome copies per milliliter of plasma within days after HCV infection. To examine whether HCV prevents or delays the recruitment of virus-specific T cells we prospectively studied blood and liver biopsies of five chimpanzees at biweekly intervals after HCV genotype 1a-infection. Two chimpanzees cleared HCV and three developed persistent infection. In all chimpanzees, CCL4, CCL5, CXCL10 and CXCL11 chemokine levels increased in blood and liver within 1-2 weeks of HCV infection. Their induction correlated closely to the induction of type I IFN (IFN-b, IFN-a2, IFN-a14, IFN-a21) and 25OAS in the liver. In vitro experiments with HCV RNA-transfected and HCV-infected hepatoma cells confirmed that HCV induced chemokines in a type I IFN-dependent manner, which could be blocked with IFN-neutralizing antibodies. However, despite the early induction of chemokines, the number of the intrahepatic lymphocytes increased not earlier than 8 weeks after infection, as shown by flow cytometry and by real-time PCR for CD8 and IFN-g mRNA. In each chimpanzee, the accumulation of CD8 lymphocytes in the liver coincided precisely with the appearance of HCV-specific, tetramer-positive CD8 T cells in the blood. HCV-specific CD8 T cells expressed chemokine receptors when they first appeared, suggesting that they were recruitable to the liver as soon as they entered the circulation. In conclusion, chemokines are induced in a type I IFN-dependent manner within 1-2 weeks after HCV infection. However, T cell recruitment and acute hepatitis depends on the appearance of HCV-specific T cells in the circulation. Thus, a delay in induction not in recruitment of HCV-specific CD8 T cells is responsible for the late onset of acute hepatitis.